USPTO Patents Application 09993647

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
30 November 2006 (30.11.2006)

PCT

(10) International Publication Number

WO 2006/125540 Al

(51) International Patent Classification:

A61K 31/4412 (2006.01) A61P 35/00 (2006.01)
A61K 31/436 (2006.01)

(21) International Application Number:

PCT/EP2006/004524

(22) International Filing Date: 13 May 2006 (13.05.2006)

(25) Filing Language:

(26) Publication Language:

(30) Priority Data:

05011477.6

English
English

27 May 2005 (27.05.2005) EP

(71) Applicant (for all designated States except US): BAYER
HEALTHCARE AG [DE/DE]; 51368 Leverkusen (DE).

(72) Inventors; and

(75) Inventors/Applicants (for US only): SCHEURING,
Urban [DE/DE]; Siegfeldstr. 15a, 53721 Siegburg (DE).
BERNARD, Ingo [DE/DE]; Auf den Rotten 46, 51789
Lindlar (DE).

(74) Common Representative: BAYER HEALTHCARE

AG; Law and Patents, Patents and Licensing, 51368
Leverkusen (DE).

(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
KG, KM, KN, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
LY, MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI,
NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG,
SK, SL, SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US,
UZ, VC, VN, YU, ZA, ZM, ZW.

(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
GN, GQ, GW, ML, MR, NE, SN, TD, TG).

Published:

— with international search report

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

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in

^ (54) Title: COMBINATION THERAPY COMPRISING A DIARYL UREA COMPOUND AND A PI3, AKT KINASE OR MTOR
INHIBITORS (RAPAMYCINS) FOR CANCER TREATMENT

(57) Abstract: The present invention relates to pharmaceutical compositions and combinations for treating cancer, comprising a
diaryl urea compound e.g. 4 {4- [3- (4-chloro-3-trifluoromethylphenyl) -ureido] -3-fluorophenoxy}-pyridine-2- carboxylia acid
methylamide and an PI3K/AKT signaling pathway inhibitor. The PI3K/AKI signaling pathway inhibitor comprises PI3 inhibitors
{like celecoxilo, viridins, wortmannins} , AKT kinase inhibitors {like perifosine, triciribine} and mTOR inhibitors {like the ra-
pamycins temsirolimus and evorolimus}.

WO 2006/125540

PCT/EP2006/004524

COMBINATION THERAPY COMPRISING A DIARYL UREA COMPOUND AND A PI3 , AKT KINASE OR
MTOR INHIBITORS (RAPAMYCINS) FOR CANCER TREATMENT

BACKGROUND OF THE INVENTION

Diaryl urea compounds e.g. 4{4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-
5 pyridine-2-carboxylic acid methylamide as described e.g. in US 20050038080 are potent anti-
cancer and anti-angiogenic agents that possess various activities, including inhibitory activity on
the VEGFR, PDGFR, raf, p38 5 and/or flt-3 kinase signalling molecules. The RAS/RAF/MEK/ERK
pathway is involved in cellular proliferation, differentiation, and transformation, and is implicated
in many cancers. The PI3K/AKT signalling pathway is another important physiological pathway
10 in cells. It mediates extracellular stimuli, including growth factors, cytokines, cell-cell adhesion
and cell-extracellular matrices (Vivanco and Sawyers, Nat Rev Cancer, 2: 489-501, 2002,
Downward, Curr Opin Cell Biol, 10: 262-267, 1998). The AKT pathway appears to be active in
many types of human cancer (Nicholson and Anderson, Cell Signal, 14: 381-395, 2002).

DESCRIPTION OF THE INVENTION

15 The present invention provides drug combinations, compositions, and methods for treating
diseases and conditions, including, but not limited to, cell proliferative disorders (such as cancer),
inflammation, immunomodulatory disorders, and conditions associated with abnormal or

*

undesirable angiogenesis. The drug combinations comprise a compound of formula I and at least
one second compound that is. an inhibitor of the PI3K/AKT signalling pathway. The methods can
20 comprise, e.g., administering a diaryl urea compound as described below and a signalling pathway
inhibitor, pharmaceutically-acceptable salts thereof, and derivatives thereof, etc.

The phosphatidylinositol-3 -kinase (PI3K) and AKT (Protein Kinase B) signalling pathway
regulates a variety of biological processes including cell survival, cell proliferation, cell growth,
and cell motility. Abnormalities in PI3K-AKT signalling contribute to the pathogenesis of a
25 number of diseases and conditions, including cell proliferative disorders (such as cancer),
inflammation, and immunomodulatory disorders.

Many growth and survival factors activate PI3K family members to specifically convert one lipid
signalling molecule, PIP2, into another, PI(3,4,5)P3. The phosphorylated product recruits Akt
family members to the inner plasma membrane, stimulating their protein kinase activity. To date,
30 many Akt effectors involved in several biological processes have been identified. For example, the
Akt kinases mediate cell survival though phosphorylation and inactivation of apoptotic machinery
components. The PI3K/AKT signalling pathway includes any members or components that

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participate in the signal transduction cascade. These include, but are not limited to, e.g., PI3-
kinase, Akt-kinase, FKBP12, mTOR (mammalian target of rapamycin; also known as FRAP,
RAFT1, or RAPT1), RAPTOR (regulatory associated protein if mTOR), TSC (tuberous sclerosis
complex), PTEN, (phosphatase and tensin homolog) and downstream effectors thereof.
5 Combinations of the present invention can be used to treat and/or prevent any condition and/or
diseases associated with any of the aforementioned activities.

An inhibitor of the PI3K/AKT signalling pathway is a compound that inhibits one or more
members of the aforementioned signal transduction cascade. While such compounds may be
referred to as pathway inhibitors, the present invention includes the use of these inhibitors to treat

10 any of the mentioned diseases or conditions, regardless of the mechanism of action or how the
therapeutic effect is achieved. Indeed, it is recognized that such compounds may have more than
one target, and the initial activity recognized for a compound may not be the activity that it
possesses in vivo when administered to a subject, or whereby it achieves its therapeutic efficacy.
Thus, the description of a compound as a pathway or protein target (e.g., Akt or mTOR) inhibitor

1 5 indicates that a compound possesses such activity, but in no way restricts a compound to having
that activity when used as a therapeutic or prophylactic agent.

Examples of AKT family members include: Aktl, Akt2 (commonly over-expressed in tumors;
Bellacosa et al., Int. J, Cancer, 64:280-285, 1995), and Akt3.

Examples of PI3K family members include: pllO-alpha, pllO-beta, pllO-delta, and pllO-gamma
20 (catalytic).

Examples of PI3K/AKT signalling pathway inhibitors include, but are not limited to, e.g., FTY720
(e.g., Lee et al., Carcinogenesis, 25(12):2397-2405, 2004);

UCN-01 (e.g., Amornphimoltham et al., Clin Cancer Res., 10(12 Pt l):4029-37, 2004).

Examples of phosphatidylinositol-3-kinase (PI3-kinase) inhibitors, include, but are not limited to,
25 e.g.,

celecoxib and analogs thereof, such as OSU-03012 and OSU-03013 (e.g., Zhu et al., Cancer Res.,
64(12):4309-18, 2004);

3-deoxy-D-myo-inositol analogs (e.g., U.S. Application No. 20040192770; Meuillet et al., OncoL
Res,, 14:513-27, 2004), such as PX-316;

WO 2006/125540 PCT/EP2006/004524

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2 '-substituted, 3'-deoxy-phosphatidyl-myo-inositol analogs (e.g., Tabellini et al., Br. J. Haematol.,
126(4):574-82, 2004);

fused heteroaryl derivatives (U.S. Pat. No. 6,608,056);

3-(imidazo[l,2-a]pyridin-3-yl) derivatives (e.g., U.S. Pat. Nos. 6,403,588 and 6,653,320);

5 Ly294002 (e.g., Vlahos, et al., J. Biol, Chem., 269(7) 5241-5248, 1994);

quinazoline-4-one derivatives, such as IC486068 (e.g., U.S. Application No. 20020161014; Geng
et al., Cancer Res., 64:4893-99, 2004);

3-(hetero)aryloxy substituted benzo(b)thiophene derivatives (e.g., WO 04 108715; also WO 04
108713);

10 viridins, including semi-synthetic viridins such as such as PX-866 (acetic acid
( 1 S,4E, 1 OR, 1 1 R, 1 3 S, 1 4R)-[4-dially laminomethy lene-6-hydroxy- 1 -methoxymethy 1- 10,13 -dime-
thy 1-3 ,7, 1 7-trioxo- 1 ,3 ,4,7, 1 0, 1 1 , 1 2, 1 3 , 1 4, 1 5 , 1 6, 1 7-dodecahy dro-2-oxa-cyclopenta[a]phenanthren-
11-yl ester) (e.g., Ihle et al., Mol Cancer Ther., 3(7):763-72, 2004; U.S. Application No.
20020037276; U.S. Pat. 5,726,167); and

15 " wortmannin and derivatives thereof (e.g., U.S. Pat. Nos. 5,504,103; 5,480^906, 5,468,773;
5,441,947; 5,378,725; 3,668,222).

Examples of Akt-kinase (also known as protein kinase B) inhibitors, include, but are not limited to,
e.g.,

Akt-1-1 (inhibits Aktl) (Barnett et al., Biochem. J., 385 (Pt.2):399-408, 2005);
20 Akt-1-1,2 (inhibits Akl and 2) (Barnett et al., Biochem. J., 385 (Pt.2):399-408, 2005);
API-59CJ-Ome (e.g., Jin et al., Br. J. Cancer., 91 :1808-12, 2004);
l-H-imidazo[4,5-c]pyridinyl compounds (e.g., WO05011700);

indole-3-carbinol and derivatives thereof (e.g., U.S. Pat. Nos. 6,656,963; Sarkar and Li, J Nutr.,
134(12 Suppl):3493S-3498S, 2004);

25 perifosine (e.g., interferes with Akt membrane localization; Dasmahapatra et al., Clin. Cancer
Res., 10(15):5242-52, 2004);

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phosphatidylinositol ether lipid analogues (e.g., Gills and Dennis, Expert, Opin. Investig. Drugs,

13:787-97, 2004);

triciribine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al., Cancer Res., 64:4394-9,

5 Examples of mTOR inhibitors include, but are not limited to, e.g.,
FKBP 12 enhancer;

rapamycins and derivatives thereof, including: CCI-779 (temsirolirnus), RAD001 (Everolimus;
WO 9409010), TAFA93 and AP23573; rapalogs, e.g. as disclosed in WO 98/02441 and WO
01/14387, e.g. AP23573, AP23464, AP23675, or AP23841; 40-(2-hydroxyethyl)rapamycin, 40-[3-

10 hydroxy(hydroxymethyl) methylpropanoate]-rapamycin (also called CC1779), 40-epi-(tetrazolyt)-
rapamycin (also called ABT578), 32-deoxorapamycin, 16-pentynyloxy-32(S)-dihydrorapamycin,
and other derivatives disclosed in WO 05005434; derivatives disclosed in USP 5,258,389, WO
94/090101, WO 92/05179, USP 5,118,677, USP 5,118,678, USP 5,100,883, USP 5,151,413, USP
5,120,842, WO 93/111130, WO 94/02136, WO 94/02485, WO 95/14023, WO 94/02136, WO

15 ' 95/16691 (e.g. SAR 943), EP 509795, WO 96/41807, WO 96/41807 and USP 5,256, 790;

- phosphorus-containing rapamycin derivatives (e.g., WO 05016252);

4H-l-benzopyran-4-one derivatives (e.g., U.S. Provisional Application No. 60/528,340).

Examples of compounds in preclinical or clinical use, include, e.g., AP23573, AP23841, CCI-779,
and RAD001.

20 Examples of phosphatidylinositol-3-kinase (PI3-kinase) inhibitors of interest are wortmannin and
the derivatives or analogs thereof and the pharmaceutical^ acceptable salts of wortmannin and its
derivatives and analogs. Consequently, methods of this invention include the use of the PI3-kinase
inhibitors of formula W:

2004).

25

(W)

wortmannin

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derivatives or analogs of the compound of formula W, pharmaceutical^ acceptable salts of the
compound of formula W, and pharmaceutical^ acceptable salts of the derivatives or analogs of the
compound of formula W.

Reference to the derivatives and analogs of wortmannin or the compound of "formula W" herein is
5 intended to include the derivatives and analogs identified in U.S. Pat. Nos. 5,504,103; 5,480,906,
5,468,773; 5,441,947; 5,378,725; 3,668,222. Suitable derivatives and analogs of the compound of
formula W include:

a) compounds of formula Wl

O

R

R'OCH 2 ., chJ

W1

1 0 . . where R is H (1 1 -desacetoxy wortmannin) or acetoxy and R' is Ci-C 6 alkyl,

b) A9,l 1- dehydrodesacetoxy wortmannin compounds of formula W2

where R' is C r C 6 alkyl,

c) 17( a-dihydro-wortmannin compounds of formula W3

R'OChL^ ch 3 V

OR

W3

where R is H or acetoxy and R' is Ci-C 6 alkyl, and R" is H, Ci-C 6 alkyl,

1 5 -C(0)OH or -C(0)0- C r C 6 alkyl;

d) open A-ring acid or ester of wortmannin compounds of formula W4

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W4

where Ri is H, methyl or ethyl and R 2 is H or methyl or

e) 1 1 -substituted and 17- substituted derivatives of wortmannin of formula W5

where R4 is =0 or -0(00)^, R 3 is =0, -OH or -0(00)^, each R* is independently phenyl,
5 Ci-C 6 alkyl or substituted Ci-C 6 alkyl, where R4 is =0 or —OH, R 3 is not =0.

The compound with the structure of formula (I) wich corresponds to 4{4-[3-(4-chloro-3-
- trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylic acid methylamide,
pharmaceutically acceptable salts, polymorphs, solvates, hydrates, metabolites and prodrugs
thereof, are collectively referred to herein as the "compounds of formula I". formula (I) is as
10 follows:

Where the plural form of the word compounds, salts, and the like, is used herein, this is taken to
mean also a single compound, salt, or the like.

The term C1-6 alkyl, unless indicated otherwise, means straight, branched chain or cyclic alkyl
15 groups having from one to six carbon atoms, which may be cyclic, linear or branched with single
or multiple branching. Such groups include for example methyl, ethyl, w-propyl, isopropyl, n-
butyl, isobutyl, sec-butyl, terr-butyl, cyclopropyl, cyclobutyl and the like. The present invention
also relates to useful forms of the compounds as disclosed herein, such as pharmaceutically

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acceptable salts and metabolites. The present invention also relatesto prodrugs of the compound of
formula (I). The term "pharmaceutical ly acceptable salt" refers to a relatively non-toxic, inorganic
or organic acid addition salt of a compound of the present invention. For example, see S. M.
Berge, et al "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19. Pharmaceutical^ acceptable
5 salts include those obtained by reacting the main compound, functioning as a base, with an
inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid,
phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic
acid and citric acid. Pharmaceutically acceptable salts also include those in which the main
compound functions as an acid and is reacted with an appropriate base to form, e.g., sodium,
10 potassium, calcium, mangnesium, ammonium, and choline salts. Those skilled in the art will
further recognize that acid addition salts of the claimed compounds may be prepared by reaction of
the compounds with the appropriate inorganic or organic acid via any of a number of known
methods. Alternatively, alkali and alkaline earth metal salts are prepared by reacting the
compounds of the invention with the appropriate base via a variety of known methods.

15 Representative salts of the compounds of this invention include the conventional non-toxic salts
and the quaternary ammonium salts which are formed, for example, from inorganic or organic
acids or bases by means well known in the art. For example, such acid addition salts include
acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
citrate, camphorate, camphorsulfonate, cinnamate, - cyclopentanepropionate, digluconate,

20 dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
itaconate, lactate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate, sulfonate, tartrate, thiocyanate, tosylate, trifluoromethanesulfonate, and undecanoate.

25 Base salts include alkali metal salts such as potassium and sodium salts, alkaline earth metal salts
such as calcium and magnesium salts, and ammonium salts with organic bases such as
dicyclohexylamine and N-methyl-D-glucamine. Additionally, basic nitrogen containing groups
may be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl
chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and

30 diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides
and iodides, aryl or aralkyl halides like benzyl and phenethyl bromides and others monosubstituted
aralkyl halides or polysubstituted aralkyl halides.

Solvates for the purposes of the invention are those forms of the compounds where solvent
molecules form a complex in the solid state and include, but are not limited to for example ethanol

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and methanol. Hydrates are a specific form of solvates, where the solvent molecule is water.

Certain pharmacologically active agents can be further modified with labile functional groups that
are cleaved after in vivo administration to furnish the parent active agent and the
pharmacologically inactive derivatizing group. These derivatives, commonly referred to as
5 prodrugs, can be used, for example, to alter the physicochemical properties of the active agent, to
target the active agent to a specific tissue, to alter the pharmacokinetic and pharmacodynamic
properties of the active agent, and to reduce undesirable side effects. Prodrugs of the invention
include, e.g., the esters of appropriate compounds of this invention that are well-tolerated,
pharmaceutically acceptable esters such as alkyl esters including methyl, ethyl, propyl, isopropyl,
10 butyl, isobutyl or pentyl esters. Additional esters such as phenyl-Ci-C 5 alkyl may be used,
although methyl ester is preferred.

Methods which can be used to synthesize other prodrugs are described in the following reviews on
the subject, which are incorporated herein by reference for their description of these synthesis
methods:

15 • Higuchi, T.; Stella, V. eds. Prodrugs As Novel Drug Delivery Systems. ACS Symposium
Series. American Chemical Society: Washington, DC (1975).

• Roche, E. B. Design of Biopharmaceutical Properties through Prodrugs and Analogs.
American Pharmaceutical Association: Washington, DC (1977).

• Sinkula, A. A.; Yalkowsky, S. H. J P harm ScL 1975, 64, 181-210.

20 • Stella, V. J.; Charman, W. N. Naringrekar, V. H. Drugs 1985, 29, 455-473.

• Bundgaard, H., ed. Design of Prodrugs. Elsevier: New York (1985).

• Stella, V. J.; Himmelstein, K. J. J. Med. Chem. 1980, 23, 1275-1282.

• Han, H-K; Amidon, G. L. AAPS Pharmsci 2000, 2, 1- 1 1 .

• Denny, W. A. Eur. J. Med. Chem. 2001, 36, 577-595.

25 • Wermuth, C. G. in Wermuth, C. G. ed. The Practice of Medicinal Chemistry Academic Press:
San Diego (1996), 697-715.

• Balant, L. P.; Doelker, E. in Wolff, M. E. ed. Burgers Medicinal Chemistry And Drug
Discovery John Wiley & Sons: New York (1997), 949-982.

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The metabolites of the compounds of this invention include oxidized derivatives of the compounds
of formula I, wherein one or more of the nitrogens are substituted with a hydroxy group; which
includes derivatives where the nitrogen atom of the pyridine group is in the oxide form, referred to
in the art as 1 -oxo-pyridine or has a hydroxy substituent, referred to in the art as 1 -hydroxy -
5 pyridine.

General Preparative Methods

The compounds of the invention may be prepared by use of known chemical reactions and
procedures as described e.g. in the following published international application WO
2005/009961.

10 The compounds of formula I have been previously characterized as having various activities,
including for inhibiting the Raf/MEK/ERK pathway, raf kinase, p38 kinase, VEGFR kinase,
PDGFR kinase. These activities and their use in treating various diseases and conditions are
disclosed in, e.g., WO 2005/009961, which are hereby incorporated by reference in their entirety.

Indications

1 5 Drug combinations of the present invention can be utilized to treat any diseases or conditions that
" are associated with, or mediated by, the cellular pathways modulated by the compounds
comprising the combinations. These pathways, include, but are not limited to signalling pathways
which comprise, e.g., VEGFR, VEGFR2, Raf/Mek/Erk, Akt/PI3K, MTOR, PTEN, etc. (see also
above). The drug combinations can be useful to treat diseases that are associated with, or

20 mediated by, mutations in one of more genes present in these pathways, including cancer-
associated mutations in PTEN, ras, Raf, Akt, PI3K, etc.

As mentioned above, although the compounds may be known as specific inhibitors, the present
invention includes any ameliorative or therapeutic effect, regardless of the mechanism of action or
how it is achieved.

25 The drug combination can have one or more of the following activities, including, anti-
proliferative; anti-tumor; anti-angiogenic; inhibiting the proliferation of endothelial or tumor cells;
anti-neoplastic; immunosuppressive; immunomodulatory; apoptosis-promoting, etc.

Conditions or diseases that can be treated in accordance with the present invention include
proliferative disorders (such as cancer), inflammatory disorders, immuno-modulatory disorders,
30 allergy, autoimmune diseases, (such as rheumatoid arthritis, or multiple sclerosis), abnormal or
excessive angiogenesis, etc.

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Any tumor or cancer can be treated, including, but not limited to, cancers having one or more
mutations in raf, VEGFR-2, VEGFR-3, PDGFR-beta, Flt-3, ras, PTEN, Akt, PI3K, mTOR, as well
as any upstream or downstream member of the signalling pathways of which they are a part. A
tumor or cancer can be treated with a drug combination of the present invention irrespective of the
5 mechanism that is responsible for it. Cancers of any organ can be treated, including cancers of, but
are not limited to, e.g., colon, pancreas, breast, prostate, bone, liver, kidney, lung, testes, skin,
pancreas, stomach, prostate, ovary, uterus, head and neck, blood cell, lymph, etc.

Cancers that can be treated in accordance with the present invention include, especially, but not
limited to, brain tumors, breast cancer, bone sarcoma (e.g., osteosarcoma and Ewings sarcoma),

10 bronchial premalignancy, endometrial cancer, glioblastoma, hematologic malignancies,
hepatocellular carcinoma, Hodgkin's disease, kidney neoplasms, leukemia, leimyosarcoma,
liposarcoma, lymphoma, Lhermitte-Duclose disease, malignant glioma, melanoma, malignant
melanoma, metastases, multiple myeloma, myeloid metaplasia, myeloplastic syndromes, non-small
cell lung cancer, pancreatic cancer, prostate cancer, renal cell carcinoma (e.g., advanced, advanced

15 refractory), rhabdomyosarcoma, soft tissue sarcoma, squamous epithelial carcinoma of the skin,
cancers associated with loss of function of PTEN; activated Akt (e.g. PTEN null tumors and
tumors with ras mutations).

Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive
lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

20 Examples of cancers of the respiratory tract include, but are not limited to, small-cell, non-small-
cell lung carcinoma, bronchial adenoma, and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to, brain stem and hypophtalmic glioma,
cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and
pineal tumor.

25 Tumors of the male reproductive organs include, but are not limited to, prostate and testicular
cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial,
cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.

Tumors of the digestive tract include, but are not limited to, anal, colon, colorectal, esophageal,
gallbladder, gastric, pancreatic, rectal, small intestine, and salivary gland cancers.

30 Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis,
ureter, and urethral cancers.

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*

Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell
carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct
carcinoma), and mixed hepatocellular cholangiocarcinoma.

5 Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma,
malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal,
and/or oropharyngeal cancers, and lip and oral cavity cancer.

Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma,
10 cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.

Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant
fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

15 In addition to inhibiting the proliferation of tumor cells, drug combinations of the present
invention can also cause tumor regression, e.g., a decrease in the size of a tumor, or in the extent of
cancer in the body.

Preference is given to the treatment of melanoma, renal cancer, hepatocellular cancer, non small
lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer or pancreatic cancer.

20 Angiogenesis-related conditions and disorders can also be treated with drug combinations of the
present invention. Inappropriate and ectopic expression of angiogenesis can be deleterious to an
organism. A number of pathological conditions are associated with the growth of extraneous
blood vessels. These include, e.g., diabetic retinopathy, neovascular glaucoma, psoriasis,
retrolental fibroplasias, angiofibroma, inflammation, restenosis, etc. In addition, the increased

25 blood supply associated with cancerous and neoplastic tissue, encourages growth, leading to rapid
tumor enlargement and metastasis. Moreover, the growth of new blood vessels in a tumor provides
an escape route for renegade cells, encouraging metastasis and the consequence spread of the
cancer.

Useful systems for modulating angiogenesis, include, e.g., neovascularization of tumor explants
30 (e.g., U.S. Pat. Nos. 5,192,744; 6,024,688), chicken chorioallantoic membrane (CAM) assay (e.g.,

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Taylor and Folkman, Nature, 297:307-312, 1982; Eliceiri et ah, J. Cell Biol., 140, 1255-1263,
1998), bovine capillary endothelial (BCE) cell assay (e.g., U.S. Pat No. 6,024,688; Polverini, P. J.
et al., Methods Enzymol., 198: 440-450, 1991), migration assays, and HUVEC (human umbilical
cord vascular endothelial cell) growth inhibition assay (e.g., U.S. Pat. No. 6,060,449). In addition,
5 useful systems for modulating lymphangiogenesis, include, e.g., rabbit ear model (e.g., Szuba et
al., FASEB J., 16(1 4): 1985-7, 2002).

Modulation of angiogenesis can be determined by any suitable method. For example, the degree
of tissue vascularity is typically determined by assessing the number and density of vesssels
present in a given sample. For example, microvessel density (MVD) can be estimated by counting

10 the number of endothelial clusters in a high-power microscopic field, or detecting a marker
specific for microvascular endothelium or other markers of growing or established blood vessels,
such as CD31 (also known as platelet-endothelial cell adhesion molecule or PECAM). A CD31
antibody can be employed in conventional immunohistological methods to immunostain tissue
sections as described by, e.g., Penfold et al., Br. J. Oral and Maxill. Surg., 34: 37-41; U.S. Pat. No.

15 6,017,949; Delias et al., Gyn. Oncol., 67:27-33, 1997; and others. Other markers for angiogenesis,
include, e.g., Vezfl (e.g., Xiang et al, Dev. Bio., 206:123-141, 1999), angiopoietin, Tie-1, and
Tie-2 (e.g., Sato et al., Nature, 376:70-74, 1995).

The drug combinations of this invention also have a broad therapeutic activity to treat or prevent
the progression of a broad array of diseases, such as inflammatory conditions, coronary restenosis,
20 tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, inflammation, certain
kidney diseases associated with proliferation of glomerular or mesangial cells, and ocular diseases
associated with retinal vessel proliferation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis,
restenosis, vascular graft restenosis, in-stent stenosis, angiogenesis, ocurlar diseases, pulmonary
fibrosis, obliterative bronchiolitis, glomerular nephritis, rheumatoid arthritis.

25 The present invention also provides for treating, preventing, modulating, etc., one or more of the
following conditions in humans and/or other mammals: retinopathy, including diabetic
retinopathy, ischemic retinal-vein occlusion, retinopathy of prematurity and age related macular
degeneration; rheumatoid arthritis, psoriasis, or bullous disorder associated with subepidermal
blister formation, including bullous pemphigoid, erythema multiforme, or dermatitis herpetiformis,

30 rheumatic fever, bone resorption, postmenopausal osteoperosis, sepsis, gram negative sepsis, septic
shock, endotoxic shock, toxic shock syndrome, systemic inflammatory response syndrome,
inflammatory bowel disease (Crohn's disease and ulcerative colitis), Jarisch-Herxheimer reaction,
asthma, adult respiratory distress syndrome, acute pulmonary fibrotic disease, pulmonary
sarcoidosis, allergic respiratory disease, silicosis, coal worker's pneumoconiosis, alveolar injury,

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hepatic failure, liver disease during acute inflammation, severe alcoholic hepatitis, malaria
(Plasmodium falciparum malaria and cerebral malaria), non-insulin-dependent diabetes mellitus
(NIDDM), congestive heart failure, damage following heart disease, atherosclerosis, Alzheimer's
disease, acute encephalitis, brain injury, multiple sclerosis (demyelation and oligodendrocyte loss
5 in multiple sclerosis), advanced cancer, lymphoid malignancy, pancreatitis, impaired wound
healing in infection, inflammation and cancer, myelodysplastic syndromes, systemic lupus
erythematosus, biliary cirrhosis, bowel necrosis, radiation injury/ toxicity following administration
of monoclonal antibodies, host-versus-graft reaction (ischemia reperfusion injury and allograft
rejections of kidney, liver, heart, and skin), lung allograft rejection (obliterative bronchitis), or

10 complications due to total hip replacement, ad an infectious disease selected from tuberculosis,
Helicobacter pylori infection during peptic ulcer disease, Chaga's disease resulting from
Trypanosoma cruzi infection, effects of Shiga-like toxin resulting from E. coli infection, effects of
enterotoxin A resulting from Staphylococcus infection, meningococcal infection, and infections
from Borrelia burgdorferi, Treponema pallidum, cytomegalovirus, influenza virus, Theiler's

15 encephalomyelitis virus, and the human immunodeficiency virus (HIV), papilloma, blastoglioma,
Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, colorectal cancer,
thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma,
- Hodgkin's disease, Burkitt's disease, arthritis, rheumatoid arthritis, diabetic retinopathy,

20 angiogenesis, restenosis, in-stent restenosis, vascular graft restenosis, pulmonary fibrosis, hepatic
cirrhosis, atherosclerosis, glomerulonephritis, diabetic nephropathy, thrombic micoangiopathy
syndromes, transplant rejection, psoriasis, diabetes, wound healing, inflammation, and
neurodegenerative diseases, hyperimmune disorders, hemangioma, myocardial angiogenesis,
coronary and cerebral collateral vascularization, ischemia, corneal disease, rubeosis, neovascular

25 glaucoma, macular degeneration retinopathy of prematurity, wound healing, ulcer Helicobacter
related diseases, fractures, endometriosis, a diabetic condition, cat scratch fever, thyroid
hyperplasia, asthma or edema following burns, trauma, chronic lung disease, stroke, polyps, cysts,
synovitis, chronic and allergic inflammation, ovarian hyperstimulation syndrome, pulmonary and
cerebral edema, keloid, fibrosis, cirrhosis, carpal tunnel syndrome, adult respiratory distress

30 syndrome, ascites, an ocular condition, a cardiovascular condition, Crow-Fukase (POEMS)
disease, Crohn's disease, glomerulonephritis, osteoarthritis, multiple sclerosis, graft rejection,
Lyme disease, sepsis, von Hippel Lindau disease, pemphigoid, Paget's disease, polycystic kidney
disease, sarcoidosis, throiditis, hyperviscosity syndrome, Osler-Weber-Rendu disease, chronic
occlusive pulmonary disease, radiation, hypoxia, preeclampsia, menometrorrhagia, endometriosis,

35 infection by Herpes simplex, ischemic retinopathy, corneal angiogenisis, Herpes Zoster, human
immunodeficiency virus, parapoxvirus, protozoa, toxoplasmosis, spondylarthritis, ankylosing

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PCT/EP2006/004524

spondylitis, Morbus Bechterew, avian influenza including e.g. serotype H5N1, and tumor-
associated effusions and edema.

The present invention provides methods of treating any of the aforementioned diseases and/or
conditions (including those mentioned in any of the cited references), comprising administering
5 effective amounts of a compound of formula I and at least one second compound that is an
inhibitor of the PI3K/AKT signalling pathway (e.g. rapamycin or a derivative or analog of
rapamycin, or wortmannin or a derivative or analog of wortmannin). An "effective amount" is the
quantity of the compound that is useful to achieve the desired result, e.g., to treat the disease or

-j

condition.

10 The present invention also relates to methods of inhibiting angiogenesis in a system comprising
cells, comprising administering to the system a combination of effective amounts of compounds
described herein. A system comprising cells can be an in vivo system, such as a tumor in a patient,
isolated organs, tissues, or cells, in vitro assays systems (CAM, BCE, etc), animal models (e.g., in
vivo, subcutaneous, cancer models), hosts in need of treatment (e.g., hosts suffering from diseases

15 having an angiogenic component, such as cancer; experiencing restenosis), etc.

In addition, the drug combinations can be administered to modulate one or more the following
. processes, cell growth (e.g., proliferation), tumor cell growth (including, e.g., differentiation, cell
survival, and/or proliferation), tumor regression, endothelial cell growth (including, e.g.,
differentiation, cell survival, and/or proliferation), angiogenesis (blood vessel growth),
20 angiogenesis, and/or hematopoiesis (e.g., proliferation, T-cell development, etc.).

Compounds or drug combinations of the present invention can be administered in any form by any
effective route, including, e.g., oral, parenteral, enteral, intravenous, intraperitoneal, topical,
transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosal,
inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and
25 intrathecal, etc. They can be administered alone, or in combination with any ingredient(s), active
or inactive. They can be administered in any effective dosage, e.g., from about 0.1 to about 200
mg/kg of total body weight.

The combinations of the present invention can be administered at any time and in any effective
form. For example, the compounds can be administered simultaneously, e.g., as a single
30 composition or dosage unit (e.g., a pill or liquid containing both compositions), or they can be
administered as separate compositions, but at the same time (e.g., where one drug is administered
intravenously and the other is administered orally or intramuscularly. The drugs can also be
administered sequentially at different times. Agents can be formulated conventionally to achieve

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PCT/EP2006/004524

the desired rates of release over extended period of times, e.g., 12-hours 5 24-hours. This can be
achieved by using agents and/or their derivatives which have suitable metabolic half-lives, and/or
by using controlled release formulations.

The drug combinations can be synergistic, e.g., where the joint action of the drugs is such that the
5 combined effect is greater than the algebraic sum of their individual effects. Thus, reduced
amounts of the drugs can be administered, e.g., reducing toxicity or other deleterious or unwanted
effects, and/or using the same amounts as used when the agents are administered alone, but
achieving greater efficacy, e.g., in having more potent antiproliferative and pro-apoptotic action.

Compounds or drug combinations of the present invention can be further combined with any other
10 suitable additive or pharmaceutical ly acceptable carrier. Such additives include any of the
substances already mentioned, as well as any of those used conventionally, such as those described
in Remington: The Science and Practice of Pharmacy (Gennaro and Gennaro, eds, 20th edition,
Lippincott Williams & Wilkins, 2000); Theory and Practice of Industrial Pharmacy (Lachman et
ah, eds., 3rd edition, Lippincott Williams & Wilkins, 1986); Encyclopedia of Pharmaceutical
15 Technology (Swarbrick and Boylan, eds., 2nd edition, Marcel Dekker, 2002). These can be
- referred to herein as "pharmaceutical^ acceptable carriers" to indicate they are combined with the
active drug and can be administered safely to a subject for therapeuticpurposes.

In addition, compounds or drug combinations of the present invention can be administered with
other active agents or therapies (e.g., radiation) that are utilized to treat any of the above-
20 mentioned diseases and/or conditions.

The present invention provides combinations of at least one compound of Formula I and at least
one second compound which is a PI3K/AKT signalling pathway inhibitor useful in treating a
disease or disorder. "Combinations" for the purposes of the invention include:

-single compositions or dosage forms which contain at least one compound of Formula I
25 and at least one second compound which is an PI3K/AKT signalling pathway inhibitor;

-combination packs containing at least one compound of Formula I and at least one second
compound which is an PI3K7AKT signalling pathway inhibitor, to be administered
concurrently or sequentially;

-kits which comprise at least one compound of Formula I and at least one second
30 compound which is an PI3K/AKT signalling pathway inhibitor packaged separate from

one another as unit dosages or as independent unit dosages, with or without instructions
that they be administered concurrently or sequentially; and

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-separate independent dosage forms of at least one compound of Formula I and at least one
second compound which is an PI3K/AKT signalling pathway inhibitor which cooperate to
achieve a therapeutic effect, e.g., prophylaxis or treatment of the same disease, when
administered concurrently or sequentially.

5 The dosage of each agent of the combination can be selected with reference to the other and/or the
type of disease and/or the disease status in order to provide the desired therapeutic activity. For
example, the active agents in the combination can be present and administered in a fixed
combination. "Fixed combination" is intended here to mean pharmaceutical forms in which the
components are present in a fixed ratio that provides the desired efficacy. These amounts can be
10 determined routinely for a particular patient, where various parameters are utilized to select the
appropriate dosage (e.g., type of cancer, age of patient, disease status, patient health, weight, etc.),
or the amounts can be relatively standard.

The combination can comprise effective amounts of at least one compound of Formula I and at
least one second compound which is a PI3K/AKT signalling pathway inhibitor, which achieves a
15 greater therapeutic efficacy than when either compound is used alone. The combination can be
. - useful to produce tumor regression, to produce disease stability, to prevent or reduce metastasis, or
other therapeutic endpoints, where the therapeutic effect is not observed when the agents are used
alone, or where an enhanced effect is observed when the combination is administered.

The relative ratios of each compound in the combination can also be selected based on their
20 respective mechanisms of action and the disease biology. For example, activating mutations of
the B-RAF gene are observed in more than 60% of human melanomas and a composition for
treatment of melanoma may advantageously comprise a formula I compound in a more potent
amount than the compound which is a P13K/AKT signalling pathway inhibitor. In comparison,
where a cancer is associated with a mutation in the PI3K/AKT signalling pathway (e.g., ovarian
25 and breast cancers), an agent which has activity in this signalling pathway can be present in more
potent amounts relative to the Ref/MEK/ERK pathway inhibitor. The relative ratios of each
compound can vary widely and this invention includes combinations for treating cancer where the
amounts of the formula I compound and the second active agent can be adjusted routinely such that
either is present in higher amounts.

30 The release of one or more agents of the combination can also be controlled, where appropriate, to
provide the desired therapeutic activity when in a single dosage form, combination pack, kit or
when in separate independent dosage forms.

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Assays

Activity of combinations of the present invention can be determined according to any effective in
vitro or in vivo method.

5 Kinase activity

Kinase activity can be determined routinely using conventional assay methods. Kinase assays

typically comprise the kinase enzyme, substrates, buffers, and components of a detection system.

A typical kinase assay involves the reaction of a protein kinase with a peptide substrate and an

it

ATP, such as P-ATP, to produce a phosphorylated end-product (for instance, a phosphoprotein
10 when a peptide substrate is used). The resulting end-product can be detected using any suitable
method. When radioactive ATP is utilized, a radioactively labeled phosphoprotein can be
separated from the unreacted gamma- P-ATP using an affinity membrane or gel electrophoresis,
and then visualized on the gel using autoradiography or detected with a scintillation counter. Non-
radioactive methods can also be used. Methods can utilize an antibody which recognizes the
15 phosphorylated substrate, e.g., an anti-phosphotyrosine antibody. For instance, kinase enzyme can
be incubated with a substrate in the presence of ATP and kinase buffer under conditions which are
effective for the enzyme to phosphorylate the substrate.. The reaction mixture can be separated,
e.g., electrophoretically, and then phosphorylation of the substrate can be measured, e.g., by
Western blotting using an anti-phosphotyrosine antibody. The antibody can be labeled with a
20 detectable label, e.g., an enzyme, such as HEP, avidin or biotin, chemiluminescent reagents, etc.
Other methods can utilize ELISA formats, affinity membrane separation, fluorescence polarization
assays, luminescent assays, etc.

An alternative to a radioactive format is time-resolved fluorescence resonance energy transfer (TR-
FRET). This method follows the standard kinase reaction, where a substrate, e.g., biotinylated

25 poly(GluTyr), is phosphorylated by a protein kinase in the presence of ATP, The end-product can
then detected with a europium chelate phosphospecific antibody (anti-phosphotyrosine or
phosphoserine/threonine), and streptavidin-APC, which binds the biotinylated substrate. These two
components are brought together spatially upon binding, and energy transfer from the
phosphospecific antibody to the acceptor (SA-APC) produces fluorescent readout in the

30 homogeneous format.

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PCT/EP2006/004524

Raf/MEK/ERK activity

A c-Raf kinase assay can be performed with a c-Raf enzyme activated (phosphorylated) by Lck
kinase. Lck-activated c-Raf (Lck/c-Raf) is produced in Sf9 insect cells by co-infecting cells with
baculoviruses expressing, under the control of the polyhedrin promoter, GST-c-Raf (from amino
5 acid 302 to amino acid 648) and Lck (full-length). Both baculoviruses are used at the multiplicity
of infection of 2.5 and the cells are harvested 48 hours post infection.

<

MEK-1 protein is produced in Sf9 insect cells by infecting cells with the baculovirus expressing
GST-MEK-1 (full-length) fusion protein at the multiplicity of infection of 5 and harvesting the
cells 48 hours post infection. Similar purification procedure is used for GST-c-Raf 302-648 and
10 GST-MEK-1.

Transfected cells are suspended at 100 mg of wet cell biomass per mL in a buffer containing 10
mM sodium phosphate, 140 mM sodium chloride pH 7.3, 0.5% Triton X-100 and the protease
inhibitor cocktail. The cells are disrupted with a Polytron homogenizer and centrifuged 30,000g
for 30 minutes. The 30,000g supernatant is applied applied onto GSH-Sepharose. The resin is
15 washed with a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100.
The GST-tagged proteins are eluted with a solution containing 100 mM Glutathione, 50 mM Tris,
_ pH 8.0, 150 mM NaCl and 0.01% Triton X-100. The purified proteins are dialyzed into a buffer
containing 20 mM Tris, pH 7.5, 1 50 mM NaCl and 20% Glycerol.

Test compounds are serially diluted in DMSO using three-fold dilutions to stock concentrations
20 ranging typically from 50 |iM to 20 nM (e.g., final concentrations in the assay can range from 1
|xM to 0.4 nM). The c-Raf biochemical assay is performed as a radioactive filtermat assay in 96-
well Costar polypropylene plates (Costar 3365). The plates are loaded with 75 |aL solution
containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng of Lck/c-Raf and 1 fig MEK-1.
Subsequently, 2 \xh of the serially diluted individual compounds is added to the reaction, prior to
25 the addition of ATP. The reaction is initiated with 25 \iL ATP solution containing 5^iM ATP and
0.3 jaCi [33P]-ATP. The plates were sealed and incubated at 32°C for 1 hour. The reaction is
quenched with the addition of 50 |il of 4 % Phosphoric Acid and harvested onto P30 filtermats
(PerkinElmer) using a Wallac Tomtec Harvester. Filtermats are washed with 1 % Phosphoric Acid
first and deinonized H20 second. The filters are dried in a microwave, soaked in scintillation fluid
30 and read in a Wallac 1205 Betaplate Counter (Wallac Inc., Atlanta, GA, U.S.A.). The results are
expressed as percent inhibition.

% Inhibition = [ 1 00-(Tib/Ti)] x 100 where

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PCT/EP2006/004524

Tib = (counts per minute with inhibitor)-(background)
Ti = (counts per minute without inhibitor)-(background)

Raf activity can also be monitored by its ability to initiate the cascade leading to ERK
phosphorylation (i.e., raf/MEK/ERK), resulting in phospho-ERK. A Bio-Plex Phospho-ERKl/2
5 immunoassay can be performed as follows:

A 96-well phospho-ERK (pERK) immunoassay, using laser flow cytometry platform has been
established to measure inhibition of basal pERK in cell lines. MDA-MB-231 cells are plated at
50,000 cells per well in 96-well microtitre plates in complete growth media. For effects of test
compounds on basal pERKl/2 inhibition, the next day after plating, MDA-MB-231 cells are

10 transferred to DMEM with 0.1% BSA and incubated with test compounds diluted 1:3 to a final
concentration of 3 mM to 12 nM in 0.1% DMSO. Cells are incubated with test compounds for 2 h,
washed, and lysed in Bio-Plex whole cell lysis buffer A. Samples are diluted with buffer B 1:1
(v/v) and directly transferred to assay plate or frozen at —80 C degrees until processed. 50 mL of
diluted MDA-MB-231 cell lysates are incubated with about 2000 of 5 micron Bio-Plex beads

15 conjugated with an anti-ERKl/2 antibody overnight on a shaker at room temperature. The next
day, biotinylated phospho-ERK 1/2 sandwich immunoassay is performed, beads are washed 3 times
> during each incubation and then 50 mL of PE-strepavidin is used as a developing reagent. The
relative fluorescence units of pERKl/2 is detected by counting 25 beads with Bio-Plex flow cell
(probe) at high sensitivity. The IC50 is calculated by taking untreated cells as maximum and no

20 cells (beads only) as background.

Phosphatidylinositol 3-kinase activity

PKI3 activity can be determined routinely, e.g., using commercially available kits (e.g., Rerkin-
Elmer, FlashPlate Platform), Frew et al., Anticancer Res., 14(6B):2425-8, 1994. See also,
publications listed under PKI3 inhibitors.

25 Akt activity

AKT can be isolated from insect cells expressing His-tagged AKT1 (aa 136-480) as described in
WO 0501 1700. Expressing cells are lysed in 25 mM HEPES, 100 mM NaCl, 20 mM imidazole;
pH 7.5 using a polytron (5 mis lysis buffer/g cells). Cell debris is removed by centrifuging at
28,000 x g for 30 minutes. The supernatant is filtered through a 4.5 micron filter then loaded onto
30 a nickel-chelating column pre-equilibrated with lysis buffer. The column is washed with 5 column
volumes (CV) of lysis buffer then with 5 CV of 20% buffer B, where buffer B is 25 mM HEPES,
100 mM NaCl, 300 mM imidazole; pH 7. His-tagged AKT1 (aa 136-480) is eluted with a 20-100%

WO 2006/125540 PCT/EP2006/004524

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linear gradient of buffer B over 10 CV. His-tagged AKTI (136-480) eluting fractions are pooled
and diluted three-fold with buffer C, where buffer C is 25 mM HEPES, pH 7. The sample is then
chromatographed over a Q-Sepharose HP column pre-equilibrated with buffer C. The column is
washed with 5 CV buffer C, then step eluted with 5 CV 10 %D, 5 CV 20%D, 5 CV 30% D, 5 CV
5 50% D. and 5 CV of 100% D; where buffer D is 25 mM HEPES, 1000 mM NaCl; pH 7.5.

His-tagged AKTI (aa 136-480) containing fractions are pooled and concentrated in a 10-kDa
molecular weight cutoff concentrator, His-tagged AKTI (aa 136-480) is chromatographed over a
Superdex 75 gel filtration column pre-equilibrated with 25 mM HEPES, 200 mM NaCl, 1 mM
DTT; pH 7.5. His-tagged AKTI (aa 136-480) fractions are examined using SDS-PAGE and mass
10 spec. The protein is pooled, concentrated, and stored at 80°C.

His-tagged AKT2 (aa 138-481) and His-tagged AKT3 (aa 135-479) can be isolated and purified in
a similar fashion.

AKT Enzyme Assay Compounds can be tested for AKT protein serine kinase inhibitory activity in
substrate phosphorylation assays. This assay examines the ability of small molecule organic

15 compounds to inhibit the serine phosphorylation of a peptide substrate. The substrate
phosphorylation assays use the catalytic domains of AKT 1, 2, or 3. AKT 17 2 and 3 are also
. commercially available from Upstate USA, Inc. The method measures the ability of the isolated
enzyme to catalyze the transfer of the gamma-phosphate from ATP onto the serine - 72 residue of a
biotinylated synthetic peptide (Biotin-ahx-ARKRERAYSFGHHA-amide). Substrate phosphory-

20 lation can be detected by the following procedure described in WO 0501 1700.

Assays are performed in 384 well U-bottom white plates. 10 nM activated AKT enzyme is
incubated for 40 minutes at room temperature in an assay volume of 20ul containing 50 mM
MOPS, pH 7.5, 20 mM MgCl 2 , 4uM ATP, 8uM peptide, 0.04 uCi [g- 33 P] ATP/well, "l mM
CHAPS, 2 mM DTT, and 1 \i\ of test compound in 100% DMSO. The reaction is stopped by the
25 addition of 50 jil SPA bead mix (Dulbecco's PBS without Mg2+ and Ca2+, 0.1 % Triton X-100, 5
mM EDTA, 50 ^M ATP, 2.5mg/ml Streptavidin-coated SPA beads). The plate is sealed, the beads
are allowed to settle overnight, and then the plate was counted in a Packard Topcount Microplate
Scintillation Counter (Packard Instrument Co., Meriden, CT).

The data for dose responses can be plotted as % Control calculated with the data reduction formula
30 100*(U1-C2)/(C1-C2) versus concentration of compound where U is the unknown value, CI is the
average control value obtained for DIVISO, and C2 is the average control value obtained for 0. 1M
EDTA. Data are fitted to the curve described by: y = ((Vmax * x) K + x)) where Vmax is the upper
asymptote and K is the IC50.

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Cell proliferation

An example of a cell proliferation assay is described in the Examples below. However,
proliferation assays can be performed by any suitable method. For example, a breast carcinoma
cell proliferation assay can be performed as follows. Other cell types can be substituted for the
, 5 MDA-MB-231 cell line.

Human breast carcinoma cells (MDA MB-231, NCI) are cultured in standard growth medium
(DMEM) supplemented with 10% heat-inactivated FBS at 37°C in 5% C0 2 (vol/ vol) in a
humidified incubator. Cells are plated at a density of 3000 cells per well in 90 (iL growth medium
in a 96 well culture dish. In order to determine T 0 h CTG values, 24 hours after plating, 100 p.L of
10 CellTiter-Glo Luminescent Reagent (Promega) is added to each well and incubated at room
temperature for 30 minutes. Luminescence is recorded on a Wallac Victor II instrument. The
CellTiter-Glo reagent results in cell lysis and generation of a luminescent signal proportional to the
amount of ATP present, which, in turn is directly proportional to the number of cells present.

Test compounds are dissolved in 100% DMSO to prepare 10 mM stocks. Stocks are further diluted
15 1 :400 in growth medium to yield working stocks of 25 ^M test compound in 0.25% DMSO. Test

compounds are serially diluted in growth medium containing 0.25% DMSO to maintain constant
- DMSO concentrations for all wells. 60 jiL of diluted test compound are added to each culture well

to give a final volume of 180 |iL. The cells with and without individual test compounds are

incubated for 72 hours at which time ATP dependent luminescence was measured, as described
20 previously, to yield T 72 h values. Optionally, the IC 50 values can be determined with a least squares

analysis program using compound concentration versus percent inhibition.

% Inhibition = [ 1 -(T 72 h test-Toh)/(T 7 2h ctrrT 0h )] * 100, where
T 7 2h test ~ ATP dependent luminescence at 72 hours in the presence of test compound
T 7 2hctri = ATP dependent luminescence at 72 hours in the absence of test compound
25 Toh — ATP dependent luminescence at Time Zero.
Angiogenesis

One useful model to study angiogenesis is based on the observation that, when a reconstituted
basement membrane matrix, such as Matrigel, supplemented with growth factor (e.g., FGF-1), is
injected subcutaneously into a host animal, endothelial cells are recruited into the matrix, forming
30 new blood vessels over a period of several days. See, e.g., Passaniti et al., Lab. Invest., 67:519-

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PCT/EP2006/004524

528, 1992. By sampling the extract at different times, angiogenesis can be temporally dissected,
permitting the identification of genes involved in all stages of angiogenesis, including, e.g.,
migration of endothelial cells into the matrix, commitment of endothelial cells to angiogenesis
pathway, cell elongation and formation of sac-like spaces, and establishment of functional
5 capillaries comprising connected, and linear structures containing red blood cells. To stabilize the
growth factor and/or slow its release from the matrix, the growth factor can be bound to heparin or
another stabilizing agent. The matrix can also be periodically re-infused with growth factor to
enhance and extend the angiogenic process.

Other useful systems for studying angiogenesis, include, e.g., neovascularization of tumor explants
10 (e.g., U.S. Pat. Nos. 5,192,744; 6,024,688), chicken chorioallantoic membrane (CAM) assay (e.g.,
. Taylor and Folkman, Nature, 297:307-312, 1982; Eliceiri et al., J. Cell Biol., 140, 1255-1263,
1998), bovine capillary endothelial (BCE) cell assay (e.g., U.S. Pat. No. 6,024,688; Polverini, P. J.
et al., Methods Enzymol., 198: 440-450, 1991), migration assays, HUVEC (human umbilical cord
vascular endothelial cell) growth inhibition assay (e.g., U.S. Pat. No. 6,060,449).

15 The present invention provides one or more of the following features.

A method of treating any of the aforementioned diseases and/or conditions, comprising
- administering effective amounts of a compound of formula I and a second compound which is an
PI3K/AKT signalling pathway inhibitor.

A method of modulating (e.g., inhibiting) one or more aforementioned activities, comprising
20 administering effective amounts of a compound of formula I and a second compound which is an
PI3K/AKT signalling pathway inhibitor.

Combinations comprising a compound of formula I and a second compound which- is an
PI3K/AKT signalling pathway inhibitor.

Without further elaboration, it is believed that one skilled in the art can, using the
25 preceding description, utilize the present invention to its fullest extent. The following
preferred specific embodiments are, therefore, to be construed as merely illustrative, and
not limitative of the remainder of the disclosure in any way whatsoever. The entire
disclosure of all patents and publications, cited above and below are hereby incorporated
by reference in their entirety.

WO 2006/125540

What we claim:

1 . A combination comprising

a compound of formula I

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PCT/EP2006/004524

(I)

5 or a pharmaceutical^ acceptable salt, polymorph, solvate, hydrate, metabolite or prodrug form
thereof, and at least one second compound which is an PI3K/AKT signalling pathway inhibitor.

2. A combination of claim 1, wherein said second compound is celecoxib, OSU-03012, OSU-
03013, PX-316, 2 '-substituted, 3'-deoxy-phosphatidyl-myo-inositol derivatives, 3-(imidazo[l,2-
a]pyridin-3-yl) derivatives, Ly294002, IC486068, 3-(hetero)aryloxy substituted benzo(b)thiophene

10 " derivatives, PX-866, perifosine, triciribine, FKBP12 enhancer, phosphatidyl inositol ether lipid
analogues, wortmannin or rapamycin or derivatives thereof, or a pharmaceutically-acceptable salt
thereof

3. A combination of claim 1, wherein said second compound is a wortmannin compound of

formula W;

15

CH 3 Q

(W)

a derivative or analog of a wortmannin compound of formula W, a pharmaceutical^ acceptable
salt of the wortmannin compound of formula W, or a pharmaceutical^ acceptable salt of the
20 derivative or analog of the wortmannin compound of formula W.

4. A combination of claim 3, wherein said derivative or analog of the formula W

is selected from

a) compounds of formula Wl

WO 2006/125540

-24-

PCT/EP2006/004524

R'OCH 2 ,, ch

W1

where R is H (1 1 -desacetoxywortmannin) or acetoxy and R' is Ci-Cealkyl,
b) A9, 11- dehydrodesacetoxywortmannin compounds of formula W2

R'OCK,,, ch

W2

where R' is Ci-C 6 alkyl,

c) 1 7(a-dihydro-wortmannin compounds of formula W3

R'OCH^ ch

OR"

W3

where R is H or acetoxy and R' is Ci-C 6 alkyl and R" is H, Ci-C 6 alkyl,

-C (O) OH or -C (O) O- C,-C 6 alkyl;

d) open A-ring acid or ester of wortmannin compounds of formula W4

W4

WO 2006/125540

PCT/EP2006/004524

-25 -

where K x is H, methyl or ethyl and R2 is H or methyl or
e) 1 1 -substituted and 17- substituted derivatives of wortmannin of formula W5

R 4

CH 3 OCH 2 ,,, ch 3 N

o

o

W5

where R4 is =0 or -0(CO)R6, R 3 is =0, -OH or -0(GO)R6, each R$ is independently
phenyl, C r C 6 alkyl or substituted C r C 6 alkyl, where R4 is =0 or -OH, R 3 is not =0.

5 5. A combination of claim 1, wherein said second compound is an Akt-kinase inhibitor.

6. A combination of claim 1, wherein said second compound is Akt-1-1, Akt-1-1,2, API-59CJ-
Ome, l-H-imidazo[4,5-c]pyridinyl derivatives, indole-3-carbinol and derivatives thereof,
perifosine, phosphatidylinositol ether lipid analogues, triciribine, or a pharmaceutically-acceptable
salt thereof.

10 7. A combination of claim 1, wherein said second compound is an mTOR inhibitor.

8. A combination of claim 1, wherein said second "compound is rapamycin, temsirolimus,

i

everolimus, AP23573, AP23675, AP23464, AP23841, 40-(2-hydroxyethyl)rapamycin, 40-[3-
hydroxy(hydroxymethyl) methylpropanoatej-rapamycin, 40-epi-(tetrazolyt)-rapamycin, 32-
deoxorapamycin, or 16-pentynyloxy-32(S)-dihydrorapamycin, SAR 943 or a pharmaceutically-
1 5 acceptable salt thereof.

9. A combination of claim 1 comprising a compound of formula (I) and wortmannin.

10. A combination of claim 1 comprising a compound of formula (I) and rapamycin.

1 1 . A combination of any of claims 1 to 1 0 wherein the amounts of the active ingredients of the
combination are synergistic.

20 12. A combination of any of claims 1 to 1 1 for treating cancer.

13. A combination of claim 12, wherein said cancer is melanoma, hepatocellular cancer, renal cell
carcinoma non small lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer
or pancreatic cancer.

WO 2006/125540

-26-

PCT/EP2006/004524

14. A method for treating cancer in a subject in need thereof comprising administering effective
amounts of a compound of formula I

9 F 3 °

or a pharmaceutical^ acceptable salt, polymorph, solvate, hydrate, metabolite or prodrug thereof,
and of a second compound which is an PI3K/AKT signalling pathway inhibitor.

15. Process for manufacturing of a combination of any of claims 1 to 1 1 for treating cancer.

16. Process of claim 15, wherein said cancer is melanoma, hepatocellular cancer, renal cell
carcinoma, non small lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer
or pancreatic cancer.

INTERNATIONAL SEARCH REPORT

A. CLASSIFICATION OF SUBJECT MATTER

INV. A61K31/4412 A61K31/436 A61P35/00

International application No

PCT/EP2006/004524

According to International Patent Classification (IPC) orto both national classification and IPC

B, FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

A61K

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

EPO-Internal , WPI Data, PAJ, CHEM ABS Data

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

x

x

Citation of document, with indication, where appropriate, of the relevant passages

US 2005/038080 Al (B0YER STEPHEN ET AL)
17 February 2005 (2005-02-17)
page 18 r-h col. example 1
page 17 [0217]

W0 2005/009961 A (BAYER PHARMACEUTICALS
CORPORATION; DUMAS, JACQUES; BOYER,
STEPHEN; RIE) 3 February 2005 (2005-02-03)
page 44, last paragraph
claim 1

□

Further documents are listed in the continuation of Box G.

* Special categories of cited documents :

"A" document defining the general state of the art which is not
considered to be of particular relevance

"E" earlier document but published on or after the international
filing date

'L" document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or
other means

■P" document published prior to the international filing date but
later than the priority date claimed

Date of the actual completion of the international search

20 July 2006

Name and mailing address of the ISA/

European Patent Office, P.B. 5618 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016

Relevant to claim No.

1-15

1—16

ID

See patent family annex.

later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention

"X" document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone

"Y" document of particular relevance; the claimed invention

cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.

document member of the same patent family

Date of mailing of the international search report

04/08/2006

Authorized officer

Baumgartner, H

Form PCT/ISA/210 (second sheet) (April 2005)

INTERNATIONAL SEARCH REPORT

Internationa] application No.

PCT/EP2006/004524

Box II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This Internationa] Search Report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

X

Claims Nos.:

because the/ relate to subject matter not required to be searched by this Authority, namely:

Although claim 14 is directed to a method of treatment of the human/animal
body, the search has been carried out and based on the alleged effects of the
compound/composition.

Claims Nos.:

because they relate to parts of the International Application that do not comply with the prescribed requirements to such
an extent that no meaningful international Search can be carried out, specifically*.

3.

Claims Nos.:

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box III Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This international Searching Authority found multiple inventions in this international application, as follows:

1.

As all required additional search fees were timely paid by the applicant, this International Search Report covers all
searchable claims.

2. | | As all searchable claims could be searched without effort Justifying an additional fee, this Authority did not invite payment
of any additional fee.

As only some of the required additional search fees were timely paid by the applicant, this International Search Report
covers only those claims for which fees were paid, specifically claims Nos.:

No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest

The additional search fees were accompanied by the applicant's protest.
No protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet (2)) (January 2004)

INTERNATIONAL SEARCH REPORT

Information on patent family members

Patent document
cited in search report

Publication
date

international application No

PCT/EP2006/004524

Patent family
members)

US 2005038080 Al

17-02-2005 NONE

WO 2005009961

03-02-2005

AR 048741 Al

AU 2004259760 Al

CA 2532865 Al

EP 1663978 A2

HR 20060073 A2

Publication
date

24-05-2006
03-02-2005
03-02-2005
07-06-2006
30-06-2006

Form PCT/ISA/210 (patent family annex) (April 2005)